Background and purpose:Differentiation of tumor tissue is an important factor on determining the prognosis of gastric cancer. This study aimed to investigate the expression levels and clinical signiifcance of gender determining region Y-box 2 (SOX2) gene and octamer binding factor 4 (OCT4) gene in gastric cancer tissues varying different differentiation degrees. Methods: Sixty cases with gastric cancer were recruited in this study. The gastric cancer tissues and corresponding normal mucosa of the 60 cases were obtained. The mRNA and protein level of SOX2, OCT4 gene are evaluated by the quantitative real-time PCR (qRT-PCR), Western blot and immunohistochemistry, respectively. The relationship between the expression levels of SOX2, OCT4 gene and clinical pathological parameters were also analyzed in this study. Results:The expression of SOX2 in both mRNA and protein levels had no signiifcant difference between the well-differentiated gastric cancer tissues and normal gastric mucosa (mRNA levels:t=0.1033, P>0.05;protein levels:t=0.116, P>0.05). However, both the mRNA and protein expression of SOX2 in patients with well-differentiated gastric cancer tissues were signiifcant higher than not only in patients with moderately differentiated gastric carcinoma (mRNA levels: t=12.48, P<0.05; protein levels: t=22.78, P<0.05) but also in patients with than poorly differentiated gastric carcinoma (mRNA levels:t=17.56, P<0.05;protein levels:t=30.00, P<0.05). In contrast to SOX2, both the mRNA and protein expression of OCT4 in patients with well-differentiated gastric cancer tissues were signiifcant lower than not only in patients with moderately differentiated gastric carcinoma (mRNA levels:t=13.23, P<0.05; protein levels: t=25.56, P<0.05) but also in patients with poorly differentiated gastric carcinoma (mRNA levels: t=12.10, P<0.05; protein levels: t=69.48, P<0.05). There was no significance of OCT4 mRNA and protein expression between the well-differentiated gastric cancer tissues and normal gastric mucosa (mRNA levels:t=2.436, P>0.05;protein levels:t=1.064, P>0.05). Immunohistochemical study demonstrated that the positive rate of SOX2 in patients with well-differentiated gastric cancer tissues (10/21) were higher than in patients with not only moderately differentiated gastric carcinoma (7/20) but also poorly differentiated gastric carcinoma (2/19, P<0.05), while the positive rate of OCT4 in cases with well-differentiated gastric cancer tissues (2/21) were lower than in cases with not only moderately differentiated gastric carcinoma (6/20) but also the poorly differentiated gastric carcinoma (12/19, P<0.05). There was no correlation between the expression of SOX2, OCT4 in gastric cancer and gender or age (P>0.05). Nevertheless, the expression of SOX2, OCT4 were positive or negative correlated with the pathological staging, the degree of inifltration and lymph node metastasis (P<0.05). Conclusion:Decreased SOX2 expression and increased expression level of OCT4 can promote the formation, development and invasion of gastric cancer and they may become biomarkers or the diagnosis, treatment and prognosis evaluation in gastric carcinoma.

Objce tive To investigate the effect and significance of down-regulation of Oct4 gene on biological characteristics of MDA-MB-231 breast cancer stem cells.Methods Breast cancer cell line MDA-MB-231 cells were used in this study.Breast cancer stem cells were isolated and enriched by serum-free culture.The obtained stem cells were identified through calculating the percentages of CD44 and CD24 stem cells by FACS and evaluating the paclitaxel resistance in vitro and tumorigenicity in mice.RT-PCR, real-time PCR (qPCR) and Western blot were used to detect Oct4 expression.RNA interference was applied to induce Oct4 down-regulation.The interference experiment set up a control group ( no siRNA transfection) , negative control group ( negative siRNA group,transfection of siRNA sequences without any interfering effect on the cells) and Oct4 siRNA group ( transfection of siRNA with interfering effect on the Oct4 gene) .Methyl thiazolyl tetrazolium ( MTT ) and Transwell chamber tests were conducted to detect the proliferation and invasion ability of MDA-MB-231 breast cancer stem cells after Oct4 knock-down, and paclitaxel inhibition test was applied to evaluate drug resistance of MDA-MB-231 breast cancer stem cells after Oct4 knock-down. Resulst MDA-MB-231 breast cancer stem cells grew as spheres cultured in serum-free suspension.MDA-MB-231 breast cancer stem cells showed a higher percentage of CD44+C/D24 -/low cells (97.2%) than that in MDA-MB-231 breast cancer cells ( 76.6%) ( P<0.05) .The tumor size in mice inoculated with MDA-MB-231 breast cancer stem cells was (124.60±13.65)mm3, significantly larger than that of mice inoculated with breast cancer cells (68.20±9.99 mm3) (P=0.0007).MDA-MB-231 breast cancer stem cells were less sensitive to paclitaxel inhibition than MDA-MB-231 breast cancer cells showing by 50% inhibitory concentration (IC50) [(4.40±0.48) μg/ml vs.(8.20±0.34) μg/ml, P<0.05].However, the expression of transcriptional factors Oct4 was higher in MDA-MB-231 breast cancer stem cells than that in breast cancer cells (P<0.05).The proliferation potential of MDA-MB-231 breast cancer stem cells with Oct4 siRNA interference was significantly lower than that in the negative siRNA and control groups ( P<0.05) from the third day.The invasion ability of MDA-MB-231 breast cancer stem cells with Oct4 siRNA interference was obviously reduced than that in the control and negative siRNA groups shown by number of penetrated cells [(46.52±2.58) vs.(79.67±3.85) and (77.29±2.13), P<0.05 for both].As for resistance to paclitaxel, IC50 of MDA-MB-231 breast cancer stem cells with Oct4siRNA interference was significantly decreased [(4.48±0.22) μg/ml] compared with that in the control [(7.99±0.59) μg/ml] and negative siRNA group [(8.10±0.68) μg/ml] (P<0.05 for both).Conclusions MDA-MB-231 breast cancer cells are successfully obtained by serum-free culture. The proliferation potential, invasion ability and drug resistance of breast cancer stem cells were down-regulated by Oct4 gene knock-down.

Objce tive To investigate the effect and significance of down-regulation of Oct4 gene on biological characteristics of MDA-MB-231 breast cancer stem cells.Methods Breast cancer cell line MDA-MB-231 cells were used in this study.Breast cancer stem cells were isolated and enriched by serum-free culture.The obtained stem cells were identified through calculating the percentages of CD44 and CD24 stem cells by FACS and evaluating the paclitaxel resistance in vitro and tumorigenicity in mice.RT-PCR, real-time PCR (qPCR) and Western blot were used to detect Oct4 expression.RNA interference was applied to induce Oct4 down-regulation.The interference experiment set up a control group ( no siRNA transfection) , negative control group ( negative siRNA group,transfection of siRNA sequences without any interfering effect on the cells) and Oct4 siRNA group ( transfection of siRNA with interfering effect on the Oct4 gene) .Methyl thiazolyl tetrazolium ( MTT ) and Transwell chamber tests were conducted to detect the proliferation and invasion ability of MDA-MB-231 breast cancer stem cells after Oct4 knock-down, and paclitaxel inhibition test was applied to evaluate drug resistance of MDA-MB-231 breast cancer stem cells after Oct4 knock-down. Resulst MDA-MB-231 breast cancer stem cells grew as spheres cultured in serum-free suspension.MDA-MB-231 breast cancer stem cells showed a higher percentage of CD44+C/D24 -/low cells (97.2%) than that in MDA-MB-231 breast cancer cells ( 76.6%) ( P<0.05) .The tumor size in mice inoculated with MDA-MB-231 breast cancer stem cells was (124.60±13.65)mm3, significantly larger than that of mice inoculated with breast cancer cells (68.20±9.99 mm3) (P=0.0007).MDA-MB-231 breast cancer stem cells were less sensitive to paclitaxel inhibition than MDA-MB-231 breast cancer cells showing by 50% inhibitory concentration (IC50) [(4.40±0.48) μg/ml vs.(8.20±0.34) μg/ml, P<0.05].However, the expression of transcriptional factors Oct4 was higher in MDA-MB-231 breast cancer stem cells than that in breast cancer cells (P<0.05).The proliferation potential of MDA-MB-231 breast cancer stem cells with Oct4 siRNA interference was significantly lower than that in the negative siRNA and control groups ( P<0.05) from the third day.The invasion ability of MDA-MB-231 breast cancer stem cells with Oct4 siRNA interference was obviously reduced than that in the control and negative siRNA groups shown by number of penetrated cells [(46.52±2.58) vs.(79.67±3.85) and (77.29±2.13), P<0.05 for both].As for resistance to paclitaxel, IC50 of MDA-MB-231 breast cancer stem cells with Oct4siRNA interference was significantly decreased [(4.48±0.22) μg/ml] compared with that in the control [(7.99±0.59) μg/ml] and negative siRNA group [(8.10±0.68) μg/ml] (P<0.05 for both).Conclusions MDA-MB-231 breast cancer cells are successfully obtained by serum-free culture. The proliferation potential, invasion ability and drug resistance of breast cancer stem cells were down-regulated by Oct4 gene knock-down.

MicroRNAs (miRNAs) are small noncoding RNAs that have a pivotal role in the post-transcriptional regulation of gene expression by sequence-specifically targeting multiple mRNAs. Although miR-33a was recently reported to play an important role in lipid homeostasis, atherosclerosis, and hepatic fibrosis, the functions of miR-33a in tumor progression and metastasis are largely unknown. Here, we found that downregulated miR-33a in breast cancer tissues correlates with lymph node metastasis. MiR-33a expression is significantly lower in the highly metastatic breast cancer cell lines than the noncancerous breast epithelial cells and non-metastatic breast cancer cells. Moreover, the overexpression of miR-33a in metastatic breast cancer cells remarkably decreases cell proliferation and invasion in vitro and significantly inhibits tumor growth and lung metastasis in vivo, whereas its knockdown in non-metastatic breast cancer cells significantly enhances cell proliferation and invasion in vitro and promotes tumor growth and lung metastasis in vivo. Combining bioinformatics prediction and biochemical analyses, we showed that ADAM9 and ROS1 are direct downstream targets of miR-33a. These findings identified miR-33a as a negative regulator of breast cancer cell proliferation and metastasis.
ADAM Proteins ; deficiency ; genetics ; Animals ; Breast Neoplasms ; genetics ; pathology ; Cell Line, Tumor ; Cell Movement ; genetics ; Cell Proliferation ; genetics ; Female ; Humans ; Lung Neoplasms ; secondary ; Membrane Proteins ; deficiency ; genetics ; Mice ; MicroRNAs ; genetics ; Middle Aged ; Neoplasm Invasiveness ; Neoplasm Metastasis ; Protein-Tyrosine Kinases ; deficiency ; genetics ; Proto-Oncogene Proteins ; deficiency ; genetics